We have previously demonstrated that H-subunit ferritin actually contributes to this process as a pro-inflammatory mediator in HSC via an iron-independent, NFkappaB-regulated signaling pathway,
inducing the expression of cytokines IL-1beta, IL-6 and RANTES. Aims: To decipher the molecular events at the level of the plasma membrane and the endocytic pathways that mediate the pro-inflammatory response of Ferritin in HSC. Methods: Primary rat HSCs were treated Fulvestrant order with 10 nM H-ferritin for 0–24 hrs. HSCs were pre-treated with inhibitors of microtubule formation (colchicine, nocodazole), lysosomal acidification (chloroquine) and intracellular protein transport (monensin), dynamin-2-dependent internalization (Dyngo-4), clathrin-coated pit (CCP) internalization (PitStop2), lipid Selleck HSP inhibitor raft/caveolae formation (beta-methyl cyclodextrin, beta-MCD) prior to
ferritin stimulation. qPCR was used to determine relative expression of Ferritin-induced transcripts. Results: Colchicine or nocodazole had no significant effect on ferritin-induced expression of IL-1beta suggesting that microtubule-dependent endocytosis is not necessary for signaling. However, inhibition
of CCP endocytosis and dynamin-dependent endocytosis in HSC totally or partially abolished Ferritin-induced pro-inflammatory response, respectively. Conversely, betaMCD–induced disruption of lipid rafts/caveoolae exacerbated response to Ferritin. Moreover, monensin treatment resulted in a 75% reduction in ferritin-induced IL-1beta expression while chloroquine completely abolished IL-1beta expression. Finally, experiments in 2-deoxyglucose-treated HSC supported that Ferritin-mediated pro-inflammatory signaling depends on glycolysis. Conclusion: These results suggest that ferritin http://www.selleck.co.jp/products/BafilomycinA1.html uptake and intracellular traffic, and therefore consequent pro-inflammatory signaling, are mediated by CCP but not via lipid rafts/caveolae. In addition, ferritin-induced HSC pro-inflammatory signaling is regulated by glycolysis linking ferritin to fibrosis in metabolic disorders. Further insights on the different cellular events that mediate ferritin-induced HSC pro-inflammatory signaling will contribute to better understanding of ferritin in the context of hepatic fibrogenesis.